ESTABLISHMENT OF A CELL LINE FROM EMBRYONIC TISSUES OF THE FLESHFLY, SARCOPHAGA PEREGRINA (INSECTA: DIPTERA)

A continuous cell line was established from embryonic tissues of the fleshfly, Sarcophaga peregrine , and was designated as NIH-SaPe-4. The primary culture was initiated in October, 1977, and the cell line was passed 68 times during the following year. The cells were heterogeneous in morphology. Most cells were diploid and their chromosomes consisted of 4 metacentric, 6 sub-metacentric and 2 micro chromosomes. The population doubling time of the cell line was about 30 hr. The cells grew faster in Mitsuhashi-Maramorosch's medium than in Schneider's medium. The cells were either stored in the usual medium at 5°C for about 3 months, or in a medium containing 10% glycerol at –80°C for a longer period. Cell growth was suppressed by 20-hydroxy-ecdysone when at a greater concentration than 0.01 μg/ml, whereas insulin showed no effects on cell growth at a strength of 0.4 and 0.04 IU/ml.     

Continuous Cell Lines from Larval Hemocytes of the Cabbage Armyworm, Mamestra brassiae

Two continuous cell lines, NIAS-MaBr-92 and NIAS-MaBr-93, were obtained from larval hemocytes of the cabbage armyworm, Mamestra brassicae . The cells grew in suspended state. Spherical cells were predominant, although there were spindle shaped and irregular shaped cells. The chromosome numbers of the cell lines varied very much with the mode of 100 of 120. The population doubling times of these cell lines were about 36hr. The cells could grow in media free of serum and lacking sterols, fatty acids and protein. The cell lines consumed ammonia and produced glutamine when cultured in MM medium. The cells could be stored at -100°C for more than two year, and at 5°C for two months. The cells were sensitive to Autographa californica nuclear polyhedrosis virus and Chilo iridescent virus.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.     

A new epithelial-like cell line from eye muscle of catla Catla catla (Hamilton): development and characterization

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major carp ( Catla catla ), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% foetal bovine serum (FBS). These cells have been subcultured >80 times over a period of 1·5 years. This cell line has been designated SICE. The SICE cell line consists predominantly of epithelial-like cells. These cells are strongly positive for epithelial markers such as pancytokeratin and cytokeratin 19. The cells were able to grow at temperatures between 25 and 32° C with optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or 20% FBS. Six marine fish viruses (fish nervous necrosis virus, marine birnavirus-NC1, chum salmon virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus-Sp and hirame rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the fourth day after subculture. Polymerase chain reaction amplification of mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence-activated cell sorter revealed that most of the cells on the second day of culture were in S-phase, indicating a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the SICE cell line can be a useful tool for transgenic and genetic manipulation studies.     

Tetrahymena Thermophila: Growth In Synthetic Nutrient Medium In the Presence and Absence of Glucose

ABSTRACT This report contains the newest directions for preparation of the synthetic nutrient medium for some Tetrahymena species that do not require lipids. In the standard medium T. thermophila. strains SB 210 and 281, multiply at 37°C with doubling times of around 2 h and at 26°C around 5 h. We have established multiplication rates as functions of variations in the composition of the medium. In media in which all components are present at one-third of the normal concentrations and only the essential amino acids are included, growth and multiplication become sharply dependent on glucose in strain SB 281. Such media may be used for selection and enrichment of certain specified cell lines.     

Effect of fatty acid supplementation on the lipid composition of Mycobacterium smegmatis ATCC 607, grown at 27° and 37°C

Mycobacterium smegmatis ATCC 607 was grown at 27 and 37°C, with and without exogenous unsaturated fatty acids, viz. elaidic, oleic and palmitoleic acids, added to the growth medium. The total lipid content of M. smegmatis ATCC 607 was lower at 27°C, and with added oleic acid, when compared with the controls, but higher in presence of palmitoleic acid. At 37°C no significant differences were noted in the total lipid content. In general, the total lipid content was lower with all of the fatty acid supplementations at both 27 and 37°C. The phosphatidylethanolamine content was slightly higher at 27°C in the presence of elaidic or palmitoleic acid, but was markedly lower with oleic acid supplementation at 37°C. The cardiolipin content was lower in the presence of any of the fatty acids at 27°C, and higher in the medium supplemented with elaidic or oleic acid at 37°C. The unsaturated to saturated fatty acids ratio was higher with palmitoleic acid supplementation at 27°C, but remained unchanged in cells grown at 37°C. The modifications in mycobacterial lipids are a reflection of the organism's ability to adapt to changing growth conditions.     

High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx mori

Abstract.  The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G₂ phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G₂ also occurs in the silkworm at high temperatures.     

STUDIES ON THE EFFECT OF TEMPERATURE ON VIRUS MULTIPLICATION IN INOCULATED LEAVES

The rate at which the Rothamsted tobacco necrosis virus (RTNV) accumulates in inoculated French bean leaves increases with rising temperature to 22°C. and then decreases. Three days after inoculation, leaves at 22°C. contain 4000 times as much virus as at 10°C. and 1000 times as much as at 30°C. At all temperatures the rate of accumulation may depend on the balance between synthesis and inactivation of RTNV, but inactivation becomes increasingly important with rise of temperature above 22° C. and as the virus content of the leaves increases. Above 22°C. the rate of multiplication may increase but less rapidly than the rate of inactivation, and exposing inoculated leaves to ultra-violet radiation at various intervals after inoculation suggests that at 30°C. RTNV multiplies in and moves from the initially infected epidermal cells in slightly less than the 6 hr. needed at 22°C. Thirty hr. are needed at 10°C. Newly formed virus is rapidly inactivated at 30°C. Raising the ambient temperature also decreases the numbers of local lesions produced by RTNV, possibly by increasing the chances that the introduced virus particles will become inactivated. Increasing the virus content of the inoculum above the level giving one lesion per sq.cm. does not increase the subsequent virus content of inoculated leaves.
At temperatures of 30°C. and below, tomato aucuba mosaic virus produces necrotic lesions in leaves of tobacco and Nicotiana glutinosa whereas above 30°C. the lesions are chlorotic. In both hosts this virus multiplies more rapidly when the infected cells are killed.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector

PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 x 10(6) cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 x 10(6) cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation.     

Free amino acid levels and the regulation of nitrate uptake in maize cell suspension cultures

The ability of individual amino acids to regulate nitrate uptakeand induction was studied in a Zea mays embryo cell line grownin suspension culture. The maize cells exhibited a marked preferencefor absorbing amino acids over nitrate when both were presentin culture medium. The addition of an individual amino acid(2 mM glutamine, glycine, aspartic acid, or arginine) to theculture medium with 1 mM nitrate completely inhibited nitrateuptake and resulted in a cycle of low levels of nitrate influxfollowed by efflux to the growth medium. Glutamine was readilyabsorbed by the cells and was particularly effective in supportingoptimum cell growth in the absence of an inorganic nitrogensource as compared to the three other amino acids evaluated.However, neither glutamine nor any of the remaining 19 proteinaceousamino acids appeared to be solely responsible for regulationof nitrate uptake and induction. The ability of amino acidsto regulate nitrate uptake and assimilation appears to be morerelated to their overall levels in the cell rather than to anaccumulation of a specific amino acid. Key words: Amino acids, nitrate uptake, maize, regulation, cell suspension culture     

Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

SOME EFFECTS OF TEMPERATURE AND OF VIRUS INHIBITORS ON INFECTION OF FRENCH-BEAN LEAVES BY RED CLOVER MOTTLE VIRUS

Keeping French-bean plants before inoculation at 36, 32 or 28°C. for 1–2 days increased their susceptibility to infection with red clover mottle virus, but longer exposures to 36 and 32°C. decreased susceptibility. Susceptibility increased most rapidly at 36°C. The number of infections was unaffected by changes in post-inoculation temperatures between 12 and 24°C., but decreased above 24°C. The rate virus multiplied increased with increase of temperature up to 28°C., but the maximum virus concentrations reached at 18, 24 and 28°C. were very similar and above the maximum reached at 30°C.
Thiouracil inhibited infection slightly but neither it nor azaguanine affected the multiplication of red clover mottle virus in French bean. Trichothecin inhibited infection and interfered with virus accumulation. Inhibition of infection was associated with macroscopic injury to the leaves, and washing leaves up to 1 hr. after inoculation prevented both inhibition and leaf damage. Virus multiplication was not resumed when leaves were transferred from trichothecin solutions to water.     

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

INHIBITION OF PROTEIN SYNTHESIS IN NONINFECTED L CELLS BY PARTIALLY PURIFIED INTERFERON PREPARATIONS

Partially purified interferon preparations, obtained from L-cell monolayers infected with Newcastle disease virus (NDV), were shown to inhibit protein synthesis in noninfected L cells. The incorporation of several amino acids-¹⁴C was equally sensitive to the pretreatment of the cells with the interferon preparation. Treatment of L-cell monolayers for 24 hr with 800 units of interferon resulted in a 50% decrease in amino acid incorporation. The degree of inhibition was found to be a function of the interferon concentration and the time of exposure of the cells to the partially purified preparations. No inhibitory effect was detected in medium obtained from noninfected cells and purified in an identical manner. The inhibitory effect was shown to be cell specific in that the partially purified interferon from L cells did not reduce amino acid incorporation in heterospecific cell lines. Heating the interferon preparations at 60°C destroyed their antiviral activity and their ability to inhibit valine-¹⁴C incorporation in L cells.     

Amino Acid Enhancement of Recovery in Dry-heat Damaged Spores of Bacillus subtilis

Spores of Bacillus subtilis MD2 and var. niger were dry-heat damaged at 150°, 160° and 170°C and recovered on media of increasing complexity. The greater the heat dose the more marked was the effect of amino acid supplements on recovery. For strain MD2 maximum germination and outgrowth of unheated spores could be obtained on a minimal salts + glucose medium with alanine, aspartic acid, glycine and methionine; the latter three amino acids served to enhance growth, not germination. The recovery of heat-damaged spores was significantly increased by adding valine plus isoleucine or arginine or glutamine. The increase was probably due to the use of valine and isoleucine as substrates of NAD-linked dehydrogenases to generate reducing power and serve as NH₃-donor, initiating germination in spores which were unable to germinate as a result of inactivation of alanine dehydrogenase. Valine or isoleucine added singly suppressed recovery by feedback inhibition of the pathways to both these amino acids during outgrowth.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Metabolically labeled cell membrane proteins in spontaneously and in SV40 virus transformed mouse fibroblasts.

A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.